GnIH基因疫苗制备及其免疫效果研究

发布时间：2018-06-08 05:51

本文选题：GnIH + RFRP-3　；参考：《华中农业大学》2017年硕士论文

【摘要】：促性腺激素抑制激素(GnIH)是动物下丘脑-垂体-性腺轴(Hypothalamic pituitary gonadal,HPG)中的下丘脑抑制激素,通过抑制Gn RH调控动物繁殖。GnIH是由十二个氨基酸组成的短肽,它通过直接或间接作用于Gn RH神经元,可降低动物机体内促性腺激素等生殖激素的分泌;同时,GnIH在性腺中也可通过自分泌/旁分泌方式调控动物的性腺机能。本研究以GnIH为靶基因构建pt S2GnIH-asd双拷贝重组质粒,在细胞水平检测重组质粒pt S2GnIH-asd的转录及表达,用构建的质粒免疫湖羊母羔,检测血清中抗体滴度水平及相关激素水平,观察母羔卵巢组织微观结构及公羊对母羔的性行为,评估GnIH基因疫苗在湖羊母羔上的免疫效果。(1)重组表达质粒pt S2GnIH-asd的构建与鉴定化学合成2对互补的GnIH基因单链核苷酸片段,通过PCR聚合获得尾端和中端GnIH目的片段,尾端GnIH片段替换p VAX-S-Gn RH-asd载体中的Gn RH基因,构建p SGnIH-asd单拷贝重组表达质粒;将中端GnIH片段插入p SGnIH-asd重组质粒中HBs Ag-S基因编码的第112至113个氨基酸密码子之间,构建p S2GnIH-asd双拷贝质粒;将含有酶切位点的t PA基因连接至p S2GnIH-asd质粒中HBs Ag-S基因的5'端,构建pt S2GnIH-asd重组表达质粒,融合目的基因记为t S2GnIH,各重组质粒经酶切、测序,其目的基因序列、插入位点及方向均正确。无内毒素提取重组表达质粒pt S2GnIH-asd及空质粒p VAX-asd,以脂质体包裹的方法转染Hela细胞,24 h后通过RT-PCR成功检测到融合基因t S2GnIH的转录;48 h后利用Western blot方法检测到融合蛋白t S2GnIH的正常表达,大小约30.91 k Da,表明在真核细胞中pt S2GnIH-asd质粒可正常进行转录和表达。(2)GnIH基因疫苗免疫湖羊母羔及免疫效果评估选取56日龄健康湖羊母羔10头,随机分为2组,疫苗组注射pt S2GnIH-asd质粒,对照组注射p VAX-asd空质粒。每组分别免疫注射3次,每次间隔21 d。初次免疫(第0 d)至第98 d期间,每14 d采血和称重,第98 d屠宰并采集组织。实验过程中母羔生长状况良好,两组母羔体重之间没有显著差异(P0.05);用Elisa检测血清抗体滴度,发现在第14 d至98 d时疫苗组母羔血清中均检测到抗GnIH抗体(P0.05);对照组LH和E2水平在整个实验期稳定,而疫苗组LH和E2水平逐步上升,在第70 d至98 d时显著高于对照组(P0.05);疫苗组血清中FSH和孕酮(P)与对照组相比没有显著差异(P0.05);疫苗组卵巢中大卵泡数显著高于对照组(P0.05);试情公羊对疫苗组持续嗅闻和追逐次数显著高于对照组(P0.05)。
[Abstract]:Gonadotropin inhibitory hormone (GnIH) is a hypothalamic inhibitory hormone in animal hypothalamic-pituitary-gonadal pituitary. GnIH is a short peptide composed of 12 amino acids that regulate animal reproduction by inhibiting GnRH. It can reduce the secretion of gonadotropin and gonadotropin by directly or indirectly acting on the GnRH neurons, and GnIH can also regulate the gonadal function by autocrine / paracrine in the gonad. In this study, the ptS2GnIH-asd double copy recombinant plasmid was constructed with GnIH as the target gene. The transcription and expression of the recombinant plasmid pt S2GnIH-asd were detected at the cell level. To observe the microstructure of ovarian tissue of female lambs and the sexual behavior of male sheep against female lambs, to evaluate the immunological effect of GnIH gene vaccine on female lambs of Hu sheep. The recombinant expression plasmid pt S2GnIH-asd was constructed and identified. Two pairs of complementary single strand nucleotides of GnIH gene were synthesized chemically. The GnIH target fragment was obtained by PCR polymerization. The GnIH fragment at the tail end replaced the GnRH gene in the pVAX-S-Gn RH-asd vector, and the single copy recombinant plasmid of pSGnIH-asd was constructed. The intermediate GnIH fragment was inserted into the 112th to 113rd amino acid codon of HBs Ag-S gene in pSGnIH-asd recombinant plasmid, and the pS2GnIH-asd double copy plasmid was constructed, and the t PA gene containing restriction site was ligated to the 5 'end of HBs Ag-S gene in pS2GnIH-asd plasmid. The recombinant expression plasmid of pt S2GnIH-asd was constructed, and the fusion gene was recorded as tS2GnIH.The recombinant plasmids were digested by enzyme and sequenced. The sequence, insertion site and direction of the target gene were correct. Recombinant expression plasmid pt S2GnIH-asd and empty plasmid pVAX-asd were extracted without endotoxin and transfected into Hela cells by liposome for 24 h. The fusion protein was successfully detected by RT-PCR for 48 h and the fusion protein was detected by Western blot. Normal expression of t S2GnIH, The size of ptS2GnIH-asd plasmid in eukaryotic cells was about 30.91 kDa, which indicated that PT S2GnIH-asd plasmid could be transcribed and expressed normally in eukaryotic cells. Ten female lambs of Hu sheep were immunized with GnIH gene vaccine and 10 female lambs were randomly divided into two groups. The vaccine group was injected with pt S2GnIH-asd plasmid. The control group was injected with pVAX-asd empty plasmid. Each group was immunized three times every 21 days. From day 0 to day 98, the blood was collected and weighed every 14 days, and the tissues were slaughtered and collected on the 98 th day. In the course of the experiment, the female lambs grew well and there was no significant difference in body weight between the two groups (P 0.05). Elisa was used to detect the titer of serum antibodies. It was found that anti-GnIH antibody P0.05 was detected in serum of female lambs from day 14 to day 98, the levels of LH and E2 in control group were stable during the whole experimental period, but the levels of LH and E2 in vaccine group increased gradually. From day 70 to day 98, it was significantly higher than that of control group (P 0.05); the serum FSH and progesterone P in vaccine group were not significantly different from those in control group; the number of large follicles in ovary of vaccine group was significantly higher than that of control group (P 0.05); And chase times were significantly higher than that of control group (P 0.05).
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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